Neurotransmitter transporters play an important role in terminating synaptic events by removing transmitters from the vicinity of pre- or postsynaptic receptors. Elucidating molecular mechanisms of neurotransmitter transporter function and modulation is of great interest to both neurobiologists and pharmacologists. It has been previously shown that ATP modulates neurotransmitter uptake. This modulatory effect may be physiologically relevant since ATP is present at high concentrations in the presynaptic vesicles and is coreleased With many neurotransmitters. However, detailed models that explain the role of ATP during neurotransmitter uptake are lacking. This proposal examines the possibility of ATP modulating directly the activity of neurotransmitter transporters. The serotonin transporter cDNA (5HTT) will be expressed in both Xenopus oocytes and mammalian cells. ATP effects on the activity of the exogenously expressed transporter will be examined by both electrophysiological recordings and substrate uptake measurements. My preliminary results show that ATP induces an inward current through the serotonin transporter. In addition, a putative ATP-binding site is found in the sequence of the serotonin transporter as well as many other neurotransmitter transporters. Thus, ATP may play a universal role in modulating transmitter uptake. Efforts will be made to characterize biophysical properties of this ATP- induced current and to correlate this current with transport activity. The putative ATP-binding site will be mutated and the effect of mutations on ATP response will be tested. In addition, the ATP effect at a real serotonergic synapse will also be examined.